M3 mAChRs are widely expressed throughout the brain. To gain insight into the role of these central M3 receptors, we recently generated mutant mice lacking M3 mAChRs only in neurons and glial cells ('brain-M3-KO mice'). Brain-M3-KO mice did not show any significant metabolic alterations, including energy expenditure and food intake. However, adult brain-M3-KO mice exhibited a dwarf-like phenotype (10% reduction in body length and 20% decrease in body weight, as compared to control mice). Consistent with this phenotype, circulating IGF-1 and growth hormone (GH) levels were significantly decreased in brain-M3-KO mice. Moreover, histological analysis revealed a marked hypoplasia of the anterior pituitary gland of brain-M3-KO mice, associated with greatly reduced pituitary GH and prolactin levels. We are currently testing the hypothesis that the lack of M3 mAChRs that normally regulate the activity of hypothalamic GHRH neurons contributes to this phenotype. These studies reveal a critical and unexpected role for neuronal M3 mAChRs in somatic growth and the proliferation of specific anterior pituitary cells.[unreadable] Central M2 and M4 mAChRs are known to play key roles in many fundamental functions of the brain, including learning and memory, locomotor activity, and mood control. For example, whole body M2 receptor KO mice displayed severe cognitive deficits in several learning and memory tasks and reduced synaptic plasticity in the hippocampus (Seeger et al. J. Neurosci. 24, 10117-27, 2004). Analysis of whole body M4 receptor KO mice showed that activation of M4 receptors counteracts the stimulatory effects of D1 dopamine receptor activation (Gomeza et al. Proc. Natl. Acad. Sci. USA 96, 10483-88, 1999). Since striatal D1 dopamine receptors play an important role in the regulation of locomotor activity and D1 receptors in the nucleus accumbens are involved in mediating the rewarding effects of drug of abuse, these findings are of considerable therapeutic interest.[unreadable] In order to learn more about the localization of the M2 and M4 mAChRs involved in these important central processes, we used Cre/loxP technology to generate a series of conditional mAChR mutant mice which lack M2 or M4 receptors only in specific regions of the brain. In the first step, we generated floxed M2 or M4 receptor mutant mice in which the receptor coding sequences are flanked by loxP sites (floxed M2 or M4 receptor mutant mice). The floxed M2 and M4 receptor mice were then crossed with transgenic mice that express Cre recombinase only in certain regions of the brain (e.g. forebrain, distinct regions of the hippocampus, D1 receptor-containing cells, etc.). The resulting mice are currently being studied by various biochemical, pharmacological, and behavioral techniques. The behavioral analysis of the mutant animals includes assays to assess learning and memory, locomotor activity, drug-seeking behavior, and several other important central functions. The analysis of the various mutant mouse strains is currently ongoing. The analysis of these newly generated mutant mice should lead to new insights into the physiological and pathophysiological roles of the M2 and M4 receptors. It is likely that these studies will identify novel therapeutic targets for the treatment of various important disorders of the CNS, including Alzheimers and Parkinsons disease and drug addiction.[unreadable] [unreadable] The following results were obtained in collaborative studies:[unreadable] [unreadable] 1. Analysis of neuronal preparations from different mAChR KO mice demonstrated that M2 and M4 receptors function as autoreceptors regulating ACh release from striatal cholinergic interneurons. Interestingly, loss of muscarinic autoreceptor function was found to impair long-term depression but not long-term potentiation in the striatum.[unreadable] (Bonsi P, et al. J. Neurosci. 28, 6258-6263, 2008)[unreadable] [unreadable] 2. Adult mice communicate by emitting ultrasonic vocalizations (USVs) during the appetitive phases of sexual behavior. Studies with different mAChR KO mice showed that M2 and M5 mAChRs are required for male USV production during male-female interactions, mostly likely via their roles in dopamine activation. These findings are important for the understanding of the neural substrates for positive affect.[unreadable] (Wang H, et al. PLoS One 3, e1893, 2008)[unreadable] [unreadable] 3. M2 receptor KO mice that had been backcrossed for 10 generations onto the C57BL/6 background showed deficits in certain learning and memory tasks, including impaired acquisition in a passive avoidance task. Moreover, M2 receptor KO mice displayed deficits in working memory in a holeboard test of spatial memory. These findings support the concept that M2 mAChRs play an important role in cognitive processes.[unreadable] (Bainbridge NK, et al. Behav. Brain Res. 190, 50-58, 2008)[unreadable] [unreadable] 4. Pharmacological studies showed that the novel compound VU10010 enhances both the affinity and efficacy of ACh at M4 receptors. Electrophysiological studies with M4 receptor KO mice demonstrated that selective potentiation of M4 receptor activity by VU10010 increases carbachol-induced depression of transmission at excitatory but not inhibitory synapses in the hippocampus, suggesting that targeting of individual mAChR subtypes could prove useful to differentially modulate hippocampal neuronal activity. [unreadable] (Shirey JK, et al. Nat. Chem. Biol. 4, 42-50, 2008)[unreadable] [unreadable] 5. Electrophysiological studies with tissues from different mAChR KO mice demonstrated that stimulation of spinal M2 and M4 receptors inhibits glycinergic inputs to spinal dorsal horn neurons. On the other hand, stimulation of spinal M3 receptors was shown to potentiate synaptic glycine release. Thus, this study revealed distinct functions for different mAChR subtypes in regulating glycinergic input to spinal dorsal horn neurons.[unreadable] (Zhang HM, et al. J. Pharmacol. Exp. Ther. 323, 963-971, 2007)